Microfluidic device

ABSTRACT

An apparatus and a method for providing one or more substance liquids to a microfluidic channel network (30). The microfluidic apparatus includes valves for switching the one or more substance liquids to a microfluidic channel network (30). The apparatus can be used to generate a sequence of the one or more substance liquids as individual droplets in an immiscible separation liquid wherein individual ones of the sequence of droplets are located between the separation liquid.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 14/344,478 filed by the present inventors on May 1, 2014, whichis U.S. national stage of PCT/EP2012/068117, which claims priority toUnited Kingdom Patent Application No. 1115895.3.

The aforementioned patent applications are hereby incorporated byreference in their entirety.

BACKGROUND OF THE INVENTION Field of the Invention

The present disclosure relates to a microfluidic apparatus and a methodfor generating a sequence of substances in a microfluidic channel. Thepresent disclosure relates in particular to a microfluidic apparatus anda method for generating a sequence of encapsulated droplets in amicrofluidic channel.

Brief Description of the Related Art

Microfluidic devices have shown great potential for many therapeutic,diagnostic, chemical or biochemical applications. They enable work witha minimized amount of substances. One aspect in microfluidics is thecontrol of flows through a channel network. Various techniques have beenestablished to generate and operate microfluidic valves. For example,multilayer soft lithography can be used to fabricate orthogonal channelsin two different layers.

Piezo electric actuators push small pins into the chip made of flexiblepolymers, thus compressing the channel and stopping the flow within. Sofar, microfluidic valves have been used mainly to generate smallcompartments and control the flow in continuous-phase devices.Droplet-based microfluidics systems allow the generation of highlymonodisperse (<3% polydispersity in terms of the volume) water-in-oil(w/o) droplets at rates of up to 10,000/s by flow-focusing a continuousaqueous phase with a second immiscible oil phase. Over the last decade,the idea of using these droplets as microreactors for parallelizedreactions within a volume range of pico- to nanoliters has beenexploited in many different applications. These include nanoparticlesynthesis, protein crystallization, single molecule PCR, proteomeanalysis, clinical diagnosis on human physiological fluids, titration ofanticoagulants and the encapsulation and screening of cells.Furthermore, several companies are commercializing droplet-basedmicrofluidics for various applications such as targeted sequencing (e.g.Raindance Technologies, GNUbio) and diagnostics (Droplet Diagnostics).

WO 2007/081386 provides a microfluidic channel for mixing andinvestigating aqueous phase droplets encapsulated in an oil stream.

A publication of Shaojiang Zeng et al. “Microvalve-actuated precisecontrol of individual droplets in microfluidic devices”, LabChip, May21, 2009; 9(10): 1340-1343 describes an example for the generation ofsequences of individual droplets separated by an immiscible oil in amicrofluidic channel. A droplet marker is described that is capable ofgenerating four different droplet species that can be fused one by onein a combinatorial fashion. While in theory this approach allows for thegeneration of many mixtures of different compounds (that can be screenedfor a desired effect or exploited for on-chip synthesis of compoundlibraries) the system has several limitations: The system is dependenton droplet fusion and only allows for the generation of combinatorialdroplet pairs; The system is driven by negative pressure. All flow isgenerated by aspirating from the outlet resulting in different dropletsizes for the different compounds when applying constant valve openingtimes. Even though this can be compensated in theory by adjusting theindividual valve opening times, only a poor level of control can beachieved. Since each infused compound needs a specific valve openingtime, it seems very challenging to systematically generate all possibledroplet pairs (and synchronize the generation of the individual dropletsto allow for pairing). In conclusion, the system can be hardly scaled up(the working principle was shown for 4 infused compounds, only, andsolely two droplet species were fused). Furthermore, a negative pressuredriven system has strict limitations in terms of the maximum flow ratesand hence the throughput.

EP 1 601 874 describes the use of mechanical devices such as aBraille-display for closing and opening valves in a microfluidic system.

It is an object of the present disclosure to overcome at least one ofthe disadvantages of prior art.

SUMMARY OF THE INVENTION

The present disclosure suggests an apparatus and a method for providinga sequence of one or more substance liquids between a separation liquid.The sequence of the one or more substance liquids may be individualdroplets in an immiscible separation liquid wherein individual ones ofthe sequence of droplets are located between the separation liquid. Theapparatus comprises a separation liquid channel; a droplet channel; anda substance liquid channel network.

In one aspect the apparatus comprises the substance liquid channelnetwork and the separation liquid channel are connected to the dropletchannel through a droplet generation junction, and wherein the substanceliquid channel network comprises a first substance liquid inlet, atleast one second substance liquid inlet and at least one substanceliquid combining area. The use of substance liquid combining area hasthe advantage that the substance can be brought together in a definedand controlled manner and the substance concentrations of the firstsubstance and at least of the second substance can be controlled. It isalso possible to combine or mix more than two substances and the numberof substances is only limited by the number of substance inlets endingin the substance liquid combining area. Only one single junction may beused for generating all droplets in the corresponding droplet channel.Droplet fusion is not necessary and can be omitted as the substances arecombined prior to droplet formation.

The disclosure also suggests a corresponding method. The methodcomprises providing a separation liquid to a separation liquid channel,providing at least one of a first substance liquid and at least onesecond substance liquid to a combination area in the substance liquidchannel network to obtain a combined substance liquid, and generatingsections of the combined substance liquid separated by the separationliquid in a droplet channel.

The substance liquid combining area may be a section of a microfluidicchannel and two or more substance liquids are guides through thecombination channel in parallel and/or in sequence. If two or moresubstance liquids are guide in parallel through the combination channel,they may flow along each other in laminar flow and mixing of thesubstance may mainly occur during or after droplet generation. However,the combination area may also comprise a mixing section wherein two ormore substances are mixed, for example by generating turbulences.

The separation liquid may be, in a water/oil system, an immiscible oiland the substance liquid may be an aqueous solution containing thesubstance or an organic solvent containing the substance or acombination of an organic solvent and an aqueous solvent. It is alsopossible to use an organic solvent/water system, wherein the separationliquid is water and the substance liquid is an organic solventcontaining the substance. Other systems can be equally used.

In an aspect, the apparatus comprises a separation liquid channel, adroplet channel, and a substance liquid channel network wherein thesubstance liquid channel network and the separation liquid channel areconnected to the droplet channel by a droplet generation junction. Thesubstance liquid channel network comprises at least one first substanceliquid inlet, at least one first valve and at least one pressure devicefor applying a continuous pressure or a continuous flow to the at leastone first substance liquid at the at least one first substance liquidinlet. The at least one first valve is switchable between a firstposition in which a liquid connection to the droplet generation junctionis open and a second position in which the liquid connection to thedroplet generation junction is closed. Applying a continuous pressure ora continuous flow to the inlet has the advantage that shorter switchingtimes and minimal perturbations occur in the system. In addition, thesize distribution of the droplets is more homogeneous and the mixing orcombination of substance liquids is better defined and controlled.

The at least one pressure device may be a pump for continuously applyinga constant flow or a pressure reservoir for continuously applying aconstant pressure. The pressure device can be used for continuouslyapplying the constant flow or constant pressure to a single one of theinlets or to several one or to all inlets in the same time.

The apparatus and the method may further comprise a first substanceliquid drain, wherein the substance liquid drain is closed in the firstposition and wherein the substance liquid drain is open in the secondposition. Using a drain or waste allows to apply a continuous flow tothe inlet and to switch the flow either to the drain or waste or towardsthe junction. Well controlled and fast switching of the liquids at thejunction can be achieved. It is possible to provide an additional drainvalve related to the drain. For example the drain valve can be opened todirect a constant flow to the drain when the first valve in in thesecond position. The drain valve may be closed, when the first valve isin the first position.

The disclosure also relates to a corresponding method comprisingproviding a separation liquid to a separation liquid channel,continuously providing at least a first substance liquid to a firstsubstance liquid channel, and switching at least on first valvedirecting the first substance liquid to a droplet generation junction,between a first position in which a liquid connection to the dropletgeneration junction is open and a second position in which the liquidconnection to the droplet generation junction is closed.

The present disclosure also suggests a method for providing a codingsequence of droplets, wherein individual ones of the sequence ofdroplets are located between a separation liquid. The method comprisesproviding a first substance liquid and at least a second substanceliquid to a microfluidic channel and selectively forming droplets of thefirst substance liquid and of the at least one second substance liquidto generate a predetermined sequence of droplets. In this way, apredetermined sequence of droplets can be generated and used as acoding. The length of the coding is technically unlimited.

Coding can be achieved by using, for example different colors or dyes inthe first substance liquid and in the second substance liquid. The dyescan be fluorescent dyes or other dyes that allow distinguishing thedroplets containing the first substance from droplets containing thesecond or any further substance. In this way a two or more color codecan be generated.

The coding may be generated in an apparatus and using method as definedabove, but the method is not limited to this.

The present disclosure also teaches an apparatus and method forproviding one or more substance liquids to a microfluidic channel. Theapparatus comprises at least one first substance liquid inlet, at leastone first valve and at least one pressure device for continuouslyapplying a constant pressure or a constant flow to the at least onefirst substance liquid at the at least one first substance liquid inlet,wherein the at least one first valve is switchable between a firstposition in which a liquid connection to the microfluidic is open and asecond position in which the liquid connection to the microfluidicchannel is closed. The method comprises continuously providing at leasta first substance liquid to a first substance liquid channel, andswitching at least one first valve directing the first substance liquidto the microfluidic channel, between a first position in which a liquidconnection to the microfluidic channel is open and a second position inwhich the liquid connection to the droplet generation junction isclosed. The method and the apparatus may be used with any type ofliquids and fluids.

The apparatus may comprise a first substance liquid drain, and thesubstance liquid drain may be closed in the first position and may beopen in the second position. This ensures that at least one way for thesubstance liquid is open at any time. A constant flow can thus beapplied to the substance liquid inlet allowing short switching timesbetween the inlets.

A person skilled in the art will understand that the above featuresrelating to several aspects of the present disclosure can be combined inany manner.

BRIEF DESCRIPTION OF THE FIGURES

Examples of the present disclosure will now be described with respect tothe accompanying Figures in which:

FIGS. 1A and 1B show a general system according to the presentdisclosure in a first configuration;

FIGS. 2A and 2B show the microfluidic channel system of FIG. 1 in asecond configuration;

FIGS. 3A and 3B show the microfluidic channel system of FIGS. 1 and 2 ina third configuration

FIG. 4 shows a second example of a microfluidic channel system of thepresent disclosure;

FIGS. 5A and 5B show possible segments of encapsulated liquid dropletsthat may be generated with the microfluidic apparatus.

FIGS. 6A, 6B and 6C show an example of combinatorial mixtures using themicrofluidic apparatus.

DETAILED DESCRIPTION

The present disclosure may be better understood with respect to examplesin which the present invention is implemented. It is to be understoodthat not all features described with respect to an example have to beimplemented and a person skilled in the art will add or remove featuresto adapt the present disclosure to specific applications orrequirements.

FIGS. 1A and 1B shows an example of a microfluidic channel network 2 inaccordance with the present disclosure. FIG. 1A shows the microfluidicchannel network 2 comprising separation liquid inlet or oil inlet 12through which a separation liquid can be inserted into a separationliquid channel 10. The separation liquid may be an immiscible oil thatcan be used for encapsulating aqueous droplets as known in the art. Theseparation liquid channel is termed oil channel 10 with respect to thedescribed example wherein an immiscible fluorinated oil was used asseparation liquid. The immiscible oil may be used as separation liquidwith aqueous and/or organic solutions as substance liquids. It is,however, also possible to use water or an aqueous solution as separationliquid, for example if the substance liquid is an organic solventcomprising the substance.

In one aspect of the invention, the separation liquid channel 10 has awidth of 185 um and a height of 52 μm. Typically the width of theseparation liquid channel 10 could vary between 150 μm and 250 μm. Theheight of the channel will be typically between 40 and 60 μm.

The oil channel 10 is connected to a junction 3 at which droplets areformed, as will be described later. A substance liquid is providedthrough a combination channel 33 from a substance liquid channel network30 to the junction 3. The combination channel 33 has a width of 100 μmin this aspect of the invention. More generally the width of thecombination channel 33 will be between 80 and 120 μm.

When a substance liquid is provided through the combination channel 33,droplets of the substance liquid are formed at the junction 3 andencapsulated by the separation liquid to form a sequence of dropletsseparated by the separation liquid. This droplet formation orencapsulation is shown in FIG. 1B in more detail. The junction 3 isconnected to a droplet channel 20, which is in the example of FIG. 1connected to a droplet liquid outlet 22. In the example shown, thejunction 3 is a T-junction which has been shown to be reliable for theformation of the droplets. Other types of junction 3 may be used aswell. The substance liquid may be an aqueous solution that may containone or more substances in different concentrations. The substance liquidis prepared and in some cases combined in the substance liquid channelnetwork as will be described in more detail below.

The droplet channel 20 may comprise an investigation area (not shown) ormay comprise further features (branches/valves) for investigating orseparating the droplets generated in the droplet channel 20. Thedroplets are generated by encapsulating droplets of an aqueous liquidgenerated in the substance liquid channel network 30. The substancechannel network 30 is combined or connected to the droplet channel 20via the combination or mixing channel 33. By providing a constant and/orcontinuous flow of oil in the oil liquid channel 10 and of at least oneaqueous substance liquid in the combination channel 33 the droplets ofthe aqueous substance liquid provided in the combination channel 33 willbe encapsulated between sections of the oil separation liquid into thedroplets that will be moved along the droplet channel 20.

The T-junction 3 is the only connection of the substance liquid channelnetwork 30 and the all of the droplets are formed from the aqueousliquid provided in the combination channel 33.

While a T-junction 3 is used in the described examples, the presentdisclosure is not limited to this type of junction, and other types ofjunctions may be equally used. For example a flow focusing junction canbe used as described for example in “Vyawahare S, Griffiths A D, MertenC A. Miniaturization and parallelization of biological and chemicalassays in microfluidic devices. Chem Biol. Oct. 29 2010;17(10):1052-1065” the content of which is incorporated by referenceherewith.

The combination channel 33 is the connection between the substanceliquid channel network 30 and the separation channel 10. The combinationchannel 33 is connected to a plurality of inlet areas 40, 41, 42, 43,44. The substance liquid channel network 30 is shown in FIGS. 1A-1B and2A-2B with five inlet areas for illustrative purposes, but the inventionis not limited to this number. It is equally possible to provide less ormore than the shown five inlet areas 40, 41, 42, 43, 44. Each of theinlet areas 40, 41, 42, 43, 44 comprises two inlets and a waste outlet.All inlet areas 40, 41, 42, 43, 44 have a similar design which will nowbe described in more detail with respect to the first inlet area 41 andthe fourth inlet area 44 illustrated in two different configurations inFIGS. 1A-1B, and FIGS. 2A-2B. FIGS. 1A-1B and 2A-2B show the sameapparatus 2 in a different configuration, i.e. with different valvesactivated.

A first inlet 411 of the first inlet area is connected to an inletchannel which leads to a first inlet or distribution valve 412 and to afirst waste valve 413. The first inlet valve 412 and the first wastevalve 413 may be activated alternatively. In the configuration shown inFIG. 1, the first inlet valve 412 is closed and the first waste valve413 is open. A liquid containing a first substance is inserted throughthe first inlet 411 and the liquid is guided via the open waste valve413 to the first waste outlet 419. The waste outlet 419 may be connectedto a waste container and a fresh liquid containing a first substance iscontinuously introduced through the first inlet 411.

In one aspect of the disclosure, the inlet channel will have a width ofaround 50 μm and more generally between 40 and 60 μm. The valves are 490μm and more generally between 440 and 550 μm. The height of the channelis 52 μm and more generally between 40 and 60 μm.

It is also possible to connect the waste outlet 419 to a reservoir andto reuse the substance liquid from the reservoir. The first substanceliquid may be continuously circulated.

While the first inlet valve 412 is closed in the configuration of FIG.1, a fourth substance liquid inserted to a fourth inlet 441 is guided tothe combination area 34 and the combination channel 22 for forming andencapsulating the droplets from the fourth substance liquid provided thefourth inlet 441 of the fourth inlet area 44. In a similar configurationto the first inlet area 41, the fourth substance liquid provided throughthe fourth inlet 441 is guided to a fourth inlet valve 442 and a fourthwaste valve 443. In the configuration illustrated in FIG. 1 the fourthinlet valve 443 is open and the fourth waste valve is close. The fourthsubstance liquid entered at the fourth inlet 441 is guided to thecombination area 34 and via the combination channel 33 to the T-junction3. The fourth liquid is formed into the droplets 6 in the dropletchannel 20 as also shown FIG. 1B.

FIGS. 2A-2B show a different configuration. The fourth inlet valve 442is now closed and the corresponding waste valve 443 is open. The fourthliquid from the fourth liquid inlet 441 will be guided to the fourthwaste 449. A continuous flow of liquid can be maintained through fourthinlet 441. In contrast, the first inlet valve 412 is now open in theconfiguration of FIGS. 2A-2B and the first waste valve 413 is closed forthe first substance liquid provided at the first inlet 411. The firstsubstance liquid is now guided to the combination area 34 and to thecombination channel 33 and droplets from the first substance liquid 7are formed in the droplet channel 20 as illustrated in FIGS. 2A-2B.

One simple application of the microfluidic apparatus 2 is described withrespect FIGS. 1A-1B and 2A-2B referring to two substance liquids, afirst substance liquid provided at first inlet 411 and the fourthsubstance liquid provided at the fourth inlet 441. It is to beunderstood that at least a second substance liquid and a third substanceliquid can be provided to the second substance liquid inlet 421 and thethird substance liquid inlet 431 and to substance liquid inlet 401.These inlets are designed in the same way and it is possible toencapsulate at least all of the four substance liquids in the channelnetwork illustrated in FIGS. 2A-2B in this way.

In addition to the first substance inlet 411, to the second substanceliquid inlet 421, the third substance liquid inlet 431 and fourthsubstance liquid inlet 441 and the substance liquid inlet 401, each ofthe inlet networks 40, 41, 42, 43, 44 comprises an additional inlet 405,415, 425, 435, 445. Each one of the additional inlets 405, 415, 425,435, 445 comprises a pair of an additional inlet valve 406, 416, 426,436, 446 and an additional waste valve 407, 417, 427, 437, 447. Forexample, the first additional inlet 415 of the first inlet area 41comprises an additional first inlet valve 416 and an additional firstwaste valve 417. By adding this additional inlets and additional valvesit is possible to use in total eight different substances at eightdifferent substance liquid inlets to the microfluidic apparatus shown inthe example of FIGS. 1A-1B, 2A-2B and 3A-3B.

In the example shown, the additional inlets 405, 415, 425, 435, 445 areconnected via the corresponding additional waste valve 407, 417, 427,437, 447 to the waste outlet 409, 419, 429, 439, 449 of thecorresponding inlet are 40, 41, 42, 43, 44, respectively. One wasteoutlet is used for two inlets. It is, however, also possible to providea separate waste outlet for each waste valve, i.e. for each inlet, forexample, if the corresponding substance liquids shall be reused. It isalso possible to combine more than two inlets to a common waste outlet.

All of the inlets are arranged in the substance liquid channel networkand are combined in the combination or mixing area 34 into a singlecombination channel 33. In this way droplets of eight differentsubstances can be encapsulated with this example. It is possible to useeach of the eight substances separately. It is also possible to combinetwo or more substances in a droplet by opening two or more of the inletvalves 402, 406, 412, 416, 422, 426, 432, 436, 442, 446 at the sametime, as shown in FIG. 3. In the example of FIG. 3, the first inletvalve 412 and the fourth inlet valve 442 are open, while all other inletvalves are closed and the substance liquid introduced at first inlet 411and at the fourth inlet 441 are combined in the combination area 34 andtransferred to the T-junction 3 via combination channel 33. A sequenceof combined droplets 8 containing the first substance and the secondsubstance can be generated.

The examples of FIGS. 1A-1B, 2A-2B and 3A-3B have been described withrespect to four and eight substance channel inlets. It is obvious to aperson skilled in the art that the disclosure is not limited to thisnumber. Many more inlets with an inlet and waste valve arrangement shownin FIGS. 1A-1B, 2A-2B and 3A-3B can be used and combined in a singlemixing area 34. An example is illustrated in FIG. 4. The example of FIG.4 shows in total sixteen substance inlet networks each with an inletchannel and an additional inlet channel allowing in this example theaddition of thirty-two substance liquids that can be combined into asequence of droplets via the single T-junction 3. The sequence ordroplets may thus contain up to 32 different droplets and all theircombinations in this example. In the example shown in FIG. 4, all valvesare in closed state which may be an initial state. It is obvious, thateach of the valves, can be individually opened to allow liquids to passto the junction 3 or to a waste as explained with the examples of FIGS.1 to 3.

It is possible to generate an unlimited number of sequence combinations.Some of these sequence combinations can be used for example for codingor for screening high number of compounds using “coding droplets”. Thesequence combinations can be used as a type of “bar code” to indicate aposition within the separation liquid. This enables, for example, anidentification of the product droplets 7 in the separation channel 10.For example, some of the droplets 7 can be used for analysis (asdescribed below) and other droplets form the identification (bar code)so that an experimenter is able to identify the product droplets 7 ofinterest.

It will be appreciated that different coding schemes can be used. Forexample single digits can be coded with one colour and tens with afurther colour. In this coding scheme the number “25” would be coded astwo droplets of a first colour followed by five droplets of a secondcolour. Alternatively a binary scheme could be used in which the firstcolour represented a 1 and the second colour represented a 0. In thiscoding scheme sample 10 (=2) would be one colour droplet followed by thesecond colour droplet and binary 101 (=5 decimal) would be a firstcolour droplet followed by the second colour droplet and finally thefirst colour droplet again.

The number of droplets can also be determined by the “length” of adroplet plug in the channel. For example, rather than generatingseparate droplets the length of the plug is determined by the time inwhich the dye is inserted into the separation channel 10 and can be readout optically. So, for example, each one of the first colour or thesecond colour can be injected for 500 ms into the separation channel 10and the length of the droplet plug formed into the separation channel 10measured.

In one aspect of the disclosure, the substance liquids making up theidentification code are inserted into the separation channel 10 at oneor more different junctions 3 at which the coding droplets are formed.This is because dyes or fluorophores that are used to form theidentification code may interfere with reactions in the product droplets7 of interest. Two different junctions 3 are used so that each junction3 is used for one single dye.

In one aspect of the invention the substance liquids for theidentification code are food dyes.

The generation of sequences is performed by activating the correspondinginlet valves. The inlet valves and the waste valves may use a Brailledisplay for activating the valves. A pin of the Braille display may bealigned with a corresponding valve and the valve can be closed bypressing the pin onto the valve to compress the channel. Brailledisplays may be preferred as they provide fast switching times of about500 ms or less. Other valves known in the art of microfluidics may beused as well.

The substance liquids are continuously supplied to the respectiveinlets, for example by pumps. The speed of the pumps or the pressureprovided does not need to be changed. Changing of the substance liquidis solely performed by switching the corresponding valves. This pressuredriven system ensures a homogeneous droplet size.

The examples have been described in a manner that at any point in timeonly one inlet valve is open at a time allowing the passage of only onesubstance liquid into the droplet channel. However, it is also possibleto open two or more valves at the same time. This will lead to a mixingof the substance liquids provided through the corresponding inlets. Thiscan be used to combine and mix different substances provided at thedifferent inlets in a predefined manner.

It is also possible to use this concept for dilution of a substance andto perform different types of assays or investigations with thedisclosed system.

EXAMPLES Example 1: Stem Cell Differentiation

The microfluidic apparatus 2 is used to screen media ingredients (e.g.growth factors and chemical stimuli) triggering the differentiation ofstem cells into specific lineages (e.g. neurons, muscle cells, etc.). Itis well known that the differentiation of stem cells is dependent onmany (chemical) factors in parallel. Hence screening combinatorialmixtures of media ingredients is required and even commerciallyexploited in conventional systems (e.g. plasticell). While aconventional setup requires large amounts of stem cells and only allowsfor relatively low throughput, the microfluidic apparatus 2 of thepresent disclosure can circumvent these limitations.

In this example, a suspension of stem cells as well as a number ofgrowth factors and chemical stimuli are continuously injected into(different inlets of) the microfluidic apparatus 2. Using a predefinedsequence of valve configurations, all possible combinations of growthfactors and chemical stimuli are co-encapsulated together with the stemcells into droplets. Downstream of the encapsulation step, the droplets6 are incubated for a time period sufficient to allow fordifferentiation of the stem cells into different lineages.

Subsequently, the droplets 6 are mixed with assay reagents (e.g.antibodies) to identify the resulting cell lineages. For this purpose,the droplets 6 containing the stem cells are fused with dropletscontaining the assay reagents before an on-chip fluorescence readout oran imaging step is performed to visualize binding of specific antibodies(indicating the differentiation into a specific lineage).

Even though all sample compositions are generated in a predefinedsequence and the order of the resulting droplets is kept constantthroughout the whole experiment, additional barcoding of the samplesmight be desired in order to enable accurate determination of thedroplets 6. This can be achieved by injecting specific fluorophores intoparticular aqueous inlets and directing their flow towards the dropmaker each time after changing the valve configurations, hencegenerating droplets forming the identification code and showing aspecific fluorescence signal. By using two different fluorophores,injecting them in an alternating fashion and varying the number ofgenerated droplets (or just the concentration of the fluorophores)sample numbers can be written in form of optical barcodes shown in FIG.5A and b.

Methodology 1: Generation of optical barcodes (large numbers) in betweenthe combinatorial samples (indicated by letters) using 2 fluorophoresinjected and encapsulated in an alternating fashion (changing thefluorophore indicates the next digit) while varying the number ofgenerated droplets. An example is shown in FIG. 5A.

Methodology 2: Generation of optical barcodes (large numbers) in betweenthe combinatorial samples (indicated by letters) using two differentfluorophores injected and encapsulated in an alternating fashion(changing the fluorophore indicates the next digit) while using tendifferent concentrations of each fluorophore. An example is shown inFIG. 5B.

Example 2: Combinatorial Chemistry

The novel microfluidic apparatus 2 is used to set up samples containingdifferent reactants for the combinatorial synthesis of bioactivemolecules. For example, the microfluidic apparatus 2 can be used to mixazides and alkenes for “click chemistry” reactions (e.g. Huisgen1,3-Dipolar Cycloaddition) in a combinatorial fashion. In this approach,a number of alkenes (n) and a number of azides (z) are continuouslyinjected into the microfluidic apparatus 2. Using a predefined sequenceof valve configurations, all possible alkeneazide pairs areco-encapsulated into the product droplets 6. Downstream of theencapsulation step, the product droplets 6 are incubated at elevatedtemperature for a time period sufficient to obtain the products of thechemical reactions.

Subsequently, the product droplets 6 are mixed with assay reagents totest for biological activity of the newly generated products. For thispurpose, the product droplets 6 are fused with droplets containing allassay reagents (e.g. a drug target and a fluorogenic substrate allowingto monitor its activity) before an on-chip fluorescence readout isperformed. The product droplets 6 combined with the assay reagentsshowing a specific fluorescence signal (e.g. particularly high/lowfluorescence intensities) indicate potent inhibition of the drug targetby a newly synthesized compound.

Even though all sample compositions are generated in a predefinedsequence and the order of the resulting droplets is kept constantthroughout the whole experiment, additional barcoding of the samplesusing coding droplets might be desired and can be achieved as describedin example 1.

Example 3: Screening Potent Drug Combinations

Many diseases cannot be cured based on the application of a single drugacting on a single drug target. For example, HIV infections are usuallytreated using highly active antiretroviral therapy (HAART). In thisapproach, drug cocktails targeting different viral proteins (e.g.Reverse Transcriptase, HIV protease or the Envelope protein) areadministered at the same time to avoid the generation of resistantmutants. Similarly, the treatment of cancer or multi-resistant bacteriaoften involves the application of drug cocktails.

The microfluidic apparatus 2 of the present disclosure can be used tosystematically encapsulate all possible combinations of a given numberof drugs into droplets and monitor their potentially cumulative effectson a co-encapsulated pathogen (also infused through one of the inlets ofthe device). After the sample generation and an incubation time allowingthe pathogen to proliferate, the product droplets 7 and their contentsare mixed with assay reagents for a viability read-out. For thispurpose, the product droplets 7 are fused with reagent dropletscontaining all assay reagents (e.g. coupling the viability of thepathogen with a fluorescence signal) before an on-chip fluorescencereadout is performed. The fused droplets showing a specific fluorescencesignal (e.g. particularly high/low fluorescence intensities) indicateefficient killing of the pathogen by a specific drug cocktail.

Optional barcoding of the samples might be desired and can be achievedas described in example 1.

Example 4: Studying Cellular Pathways Using Chemical Perturbations

The microfluidic apparatus 2 of the present disclosure is used toanalyze and map cellular pathways and/or interactions of differentcellular factors (e.g. proteins). For this purpose, the pathways areperturbed using combinations of known inhibitory or stimulatingcompounds (in regard to a certain phenotype). Subsequently a readout isperformed to analyze if the combination of compounds results incumulative, saturated or competitive effects (compared to the effect ofindividual compounds). For example, if the combination of two inhibitorsdoes not mediate stronger inhibition than each of the two inhibitorsindividually, it seems highly likely that their targets are involved inthe same pathway. In contrast, in case a cumulative (increased) effectis observed, it is more likely that the targets are involved in twodifferent pathways that are not directly linked. Systematic screening ofcombinations of inhibitors and stimuli hence allows to derive detailedinteraction maps of cellular pathways.

The microfluidic apparatus 2 of the present disclosure can be used tosystematically encapsulate all possible combinations of a set ofdifferent inhibitors and stimuli into droplets and monitor their effectson co-encapsulated cells (also infused through one of the inlets of thedevice). After their generation, the samples in the product droplets 7are incubated to allow for inhibition or stimulation of theircorresponding cellular targets. Subsequently, the contents of theproduct droplets 7 are mixed with assay reagents allowing to monitor thephenotype. For example, the product droplets 7 are fused with reagentdroplets containing all assay reagents (e.g. coupling the inhibition ofcellular pathways with a fluorescence signal) before an on-chipfluorescence readout is performed. Quantitative readout of thefluorescence signals allows determination if the combination ofcompounds results in cumulative, saturated or competitive effects andhence enables to derive a detailed interaction map (regarding thetargets of the compounds).

Optional barcoding of the samples might be desired and can be achievedas described in example 1.

It is apparent to a person skilled in the art that it is not necessaryto implement all features of the present disclosure. For example it ispossible to provide only one or two inlets. It is also possible toprovide a plurality of T-junctions connected to a plurality of substanceliquid channel networks.

Example 5: Combinatorial Mixtures

The microfluidic apparatus of the disclosure can be used to generatecombinatorial mixtures. An example is shown in FIG. 6A-c in which fourdifferent concentrations of a fluorescent dye are used as the substanceliquids. FIG. 6A shows on the x-axis the concentrations of the dye (25μm, 50 μm, 100 μm and 200 μm) and on the y-axis the degree offluorescence in arbitrary units (AU). A regression line was fitted tothe data and was best represented by the equation y=0.0028x-0.1033.

FIG. 6B shows the result of mixing various combinations of two of theconcentrations of the dye in approximately equal amounts. The squaresshow the theoretical values calculated from the equation and the rhombishow the actual measured results. Similarly FIG. 6C shows the results ofmixing three of the concentrations of the die in approximately equalamounts. The experimental results are substantially in agreement withthe theoretical results calculated. This shows that combinatorial mixingcan take place in the microfluidic channel network 30.

Example 6: Combination with Microscope

It is further possible to combine the droplet channel 20 with amicroscope or other investigation or experimental setups to investigatethe compounds generated in the droplet sequence. The droplet sequencemay also be used for further experiments and may be applied tobiological and non-biological systems arranged inside the micro channelnetwork.

What is claimed is:
 1. A method for providing a sequence of droplets ofone or more substance liquids between a separation liquid, the methodcomprising: providing a separation liquid to a separation liquidchannel; providing at least one of a first substance liquid from a firstsubstance liquid inlet to a combination area and at least one secondsubstance liquid from a second substance liquid inlet to said tocombination area in the substance liquid channel network to obtain acombined substance liquid; and generating sections of the combinedsubstance liquid separated by the separation liquid in a dropletchannel, switching at least one first inlet valve between a firstposition, in which a liquid connection from the at least one firstsubstance liquid inlet to the combining area microfluidic channelnetwork is open, and a second position, in which the liquid connectionfrom the at least one first substance liquid inlet to substance liquidcombining area the microfluidic channel network is closed, switching asecond inlet valve between a first position, in which a liquidconnection from the second substance liquid inlet to the substanceliquid combining area microfluidic channel network is open, and a secondposition, in which the liquid connection from the second substanceliquid inlet to substance liquid combining area the microfluidic channelnetwork is closed.
 2. The method of claim 1, wherein the providing atleast one of a first substance liquid and at least one second substanceliquid to a mixing area comprises supplying the first substance liquidcontinuously to a first substance liquid inlet and supplying the atleast one second substance liquid continuously to a second substanceliquid inlet.
 3. A method for providing a sequence of droplets of one ormore substance liquids between a separation liquid, the methodcomprising: providing a separation liquid to a separation liquidchannel; continuously providing at least a first substance liquid to afirst substance liquid inlet; continuously providing a second substanceliquid to a second substance liquid inlet and switching at least onefirst valve directing the first substance liquid to a droplet generationjunction at which droplets are formed, between a first position in whicha liquid connection to the droplet generation junction is open and asecond position in which the liquid connection to the droplet generationjunction is closed, switching a second inlet valve directing the secondsubstance liquid to the droplet generation junction at which dropletsare formed, between a first position in which a liquid connection to thedroplet generation junction is open and a second position in which theliquid connection to the droplet generation junction is closed whereinthe switching of the first inlet valve and of the second inlet valve isadapted to modify one or more liquid substances in the droplet.
 4. Themethod of claim 3, wherein the continuously providing of the at leastone first substance liquid to the first substance liquid inlet comprisesapplying a continuous pressure.
 5. The method of claim 3, wherein thecontinuously providing of the at least one first substance liquid to thefirst substance liquid inlet comprises applying at least one of aconstant pressure or a constant flow of the at least one first substanceliquid to the first substance liquid inlet.
 6. The method of claim 3,wherein the switching of the at least one first valve comprisesswitching between the first position in which a substance liquid drainis closed and the liquid connection to the droplet generation junctionis open and the second position in which the substance liquid drain isopen and the liquid connection to the droplet generation junction isclosed.